This project is aimed at elucidation of the structural relationships of biotin-containing enzymes. This is a relatively small group of enzymes with specialized metabolic functions. In general, the enzymes are large, multi-subunit proteins with complex structural and regulatory properties. The studies have involved mainly methylmalonyl-CoA-oxalacetate transcarboxylase, an enzyme from the propionic acid bacteria that transfers carboxyl groups and pyruvate carboxylase, an enzyme from diverse sources that catalyzes the formation of oxalacetate. Previous studies on transcarboxylase have shown that this enzyme may consist of as many as 30 polypeptide chains although only three types of chains are involved. Plans call for the sequencing of the smaller peptide which contains the biotin-moiety in order to compare this structure with analogous portions of other biotin enzymes. Also, further experiments will be designed to elucidate the relationship of the central unit of this enzyme to its 1 to 6 peripheral subunits. Pyruvate carboxylases from animal tissues appear to be cyclic tetramers with spherical subunits arranged at the corners of a square. The enzymes from yeast and some species of bacteria have a more complex structure and will be investigated by cross-linking, electron microscopic and physical means. Affinity labeling procedures will be used to define the catalytic and activation sites of this group of enzymes. BIBLIOGRAPHIC REFERENCES: Barritt, G.J., Zander, G.L. and Utter, M.F. in "Gluconeogenesis" Eds. Hanson and Mehlman. John Wiley and Sons (1976) pp. 3-46. The Regulation of Pyruvate Carboxylase Activity in Gluconeogenic Tissues. Barden, R.E., Taylor, B.L., Isohashi, F., Frey, W.H., Zander, G., Lee, J.C. and Utter, M.F. Proc. Nat. Acad. Sci. 72, 4308-4312 (1976). Structural Properties of Pyruvate Carboxylases from Chicken Liver and Other Sources.